With the explosive increase in genome sequencing, there appear to be an unprecedented number of bacteria under investigation at the molecular level;however, we need to exercise caution against over-interpretation of conserved genome contents. Vast sectors of our understanding of basic biological processes remain derived from a very small number of model organisms. Although orthologous genes can be identified with increasing reliability and ease, conserved function and conserved regulation are still assumptions, and little is known about the evolutionary dynamics underlying divergence of regulation in prokaryotes. In the previous period of this award, we successfully developed and applied multiple genome alignment tools to characterize the rates and patterns of genome evolution among enterobacteria and to delineate conserved and lineage-specific regions of genomes. Here, we continue dissecting the evolutionary history of this group of biomedically and agriculturally significant bacteria, extending our analyses to examine 1) the extent to which orthologous genes are regulated by orthologous regulators using orthologous binding sites, and 2) the integration of horizontally transferred genes into global regulons. The transcriptional response to growth in an oxygen- limited environment will serve as a case study of regulatory network evolution across the enterobacteria. We have selected this condition because it is well characterized in one model system, E. coli K-12, the transcriptional response involves a substantial number of genes, and metabolism in the presence or absence of oxygen plays a key role in defining environments permissible for bacterial growth such as pathogen survival in specific host environments. RELEVANCE: Pathogenic bacteria must regulate the expression of their genes during progressive stages of infection in response to a wide variety of external and internal environmental cues. Using genome sequence comparisons and experiments that dissect the response to varying concentrations of oxygen, an important factor in the host environment, we will determine the rates and patterns of regulatory system evolution across a group of biomedically and agriculturally significant bacteria. This will improve general models of bacterial regulatory evolution and provide specific insights into virulence factors regulated in an oxygen-dependent manner.